LeBauer:Protocol/Enzyme

Overview

Enzyme assays for litter and soil.

Adapted from

• Steven Allison 2008 (Allison Lab),
  • Steve's Protocol
• Bob Sinsabaugh 1994 (Center for Dead Plant Studies)

Materials

Solutions

• 1.0 M NaOH Sodium hydroxide
  • 4g NaOH pellets
  • 100mL DI water

• 50 mM sodium acetate buffer (for 1 L)
  • 4.37 g Sodium acetate anhydrous (CAS 127-09-3)
  • 1.1 ml glacial acetic acid (CAS 64-19-7)
  • fill 1000 ml volumetric flask
  • titrate solution to pH = 5 with additional acetic acid

Substrates

PPO polyphenol oxidase

• 50 mM pyrogallolX μL reagent 2
  • 631 mg pyrogallol (CAS 87-66-1)
  • 1.861 g disodium dihydrate EDTA
  • 100 ml buffer
• 100 ml volumetric flask

BG: [math]\displaystyle{ \beta }[/math]-glucosidase

• 5 mM p-Np-[math]\displaystyle{ \beta }[/math]-gucopyranoside
  • 150.7 mg substrate
  • 100 ml buffer
• 100 ml volumetric flask

Procedure

Preparing soil sample:

1. Add 10 ml sodium acetate buffer into each soil tube. Shake gently.
2. Place tubes on plate shaker for 1 hour on speed 180 for mixing.

Preparing assay trays:

1. Rinse trays free from any contaminates or debris with sodium acetate buffer. Rinse trays out again with ethanol. Place upside down to dry.
2. Label one set of trays BG (A-H) and another set PPO (A-H)
3. See figure on right for arrangement of a 96 well plate with 7 reps each of 6 samples per tray.

Combining enzymes and substrates:

1. Add 200μl buffer to blank wells
2. Add 150μl buffer to homogenate control wells
3. Add 50μl buffer to substrate control wells
4. Add 50 µl homogenate to the homogenate control and assay wells.
5. Add 150 µl substrate to the substrate control and assay wells.

See [2] for more details. Analysis on spectrometer follows Allison's procedure.

Notes

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References

Relevant papers

1. Allison, S.D., and Jastrow, J.D. 2006. Activities of extracellular enzymes in physically isolated fractions of restored grassland soils DOI:10.1016/j.soilbio.2006.04.011
2. LeBauer, D.S. 2010. Litter degradation rate and beta-glucosidase activity increase with fungal diversity. Canadian Journal of Forest Research. 40(6): 1076–1085 DOI:10.1139/X10-054